S311-08

SOD Assay Kit-WST #Unit: 100 tests

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Description:

Storage: 0-5 ºC

Shipping Condition: Blue Ice or Ambient Temperature

Chemical Name:

CAS Number:

Advantages:
- Only kit to measure 100% inhibition by SOD
- pH independent IC₅₀ determination
- Convenient 96-well microplate colorimetric assay
- Low-background noise measurement

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion (O₂·^-) into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. In mammals, cytosolic SOD has a greenish color and consists of two subunits: Each subunit contains copper and zinc (Cu/Zn-SOD). Mitochondrial and bacterial SOD has a reddish-purple color and contains manganese (Mn-SOD). E. coli has Mn-SOD and Fe-SOD.

Several direct and indirect methods have been developed to determine SOD activity. An indirect method using nitrotetrazolium blue is often used because of its convenience. However, there are several disadvantages to this method, such as poor water solubility of the formazan dye and its reaction with the reduced form of xanthine oxidase. Though cytochrome C is also commonly used for SOD activity detection, its reactivity with superoxide is too high to determine low levels of SOD activity.

SOD Assay Kit-WST allows very convenient and highly sensitive SOD assay by utilizing Dojindo's highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with a superoxide anion. WST-1 is 70 times less reactive with superoxide anion than cytochrome C; therefore, highly sensitive SOD detection is possible and samples can be diluted with buffer to minimize background problems. WST-1 does not react with the reduced form of xanthine oxidase; therefore, even 100% inhibition with SOD is detectable. The rate of WST-1 reduction by superoxide anion is linearly related to the xanthine oxidase activity, and is inhibited by SOD (see figure below). Therefore, the IC₅₀ (50% inhibition concentration) of SOD or SOD-like materials can be determined using colorimetric methods (patent filing).

Inhibition Curve Prepared Using SOD from Bovine Erythrocytes
SOD Inhibition Curve

Mechanism of SOD Assay
SOD Assay Kit-WST

Preparation of Sample Solution

Cells(Adherent cells: 9x106 cells, Leukocytes: 1.2x107)
1. Harvest cells with a scraper, centrifuge at 2,000 g for 10 min at 4ºC, and discard the supernatant.
2. Wash the cells with 1 ml PBS, and centrifuge at 2,000 g for 10 min at 4ºC. Discard the supernatant. Repeat this step.
3. Break cells using the freeze-thaw method (-20ºC for 20 min, then 37ºC bath 10 min, repeat twice).
4. Add 1 ml of PBS. If necessary, sonicate the cell lysate on an ice bath (60 W with 0.5 sec interval for 15 min).
5. Centrifuge at 10,000 g for 15 min at 4ºC.
6. Remove the supernatant and dilute it with PBS to prepare sample solution.

　
Tissue(100 mg)
1. Wash the tissue with saline to remove as much blood as possible. Blot the tissue with paper towels and then measure its weight.
2. Add 400-900 µl sucrose buffer (0.25 M sucrose, 10 mM Tris, 1 mM EDTA, pH 7.4) and homogenize the sample using Teflon homogenizer. If necessary, sonicate the homogenized sample on an ice bath (60W with 0.5 second intervals for 15 min).
3. Centrifuge the homogenized sample at 10,000 g for 15 min at 4ºC, and transfer the supernatant to a new tube.
4. Dilute the supernatant with distilled water to prepare sample solution.

　
Erythrocytes or Serum
1. Centrifuge 2-3 ml of anticoagulant-treated blood (such as heparin 10 U/ml final concentration) at 600 g for 10 min at 4ºC.
2. Remove the supernatant and dilute it with saline to use as a plasma sample. Add saline to the pellet to prepare the same volume and suspend the pellet.
3. Centrifuge the pellet suspension at 600 g for 10 min at 4ºC and discard the supernatant.
4. Add the same volume of saline and repeat Step 3 twice.
5. Suspend the pellet with 4 ml distilled water, then add 1 ml ethanol and 0.6 ml chloroform.
6. Shake the mixture vigorously with a shaker for 15 min at 4ºC.
7. Centrifuge the mixture at 600 g for 10 min at 4ºC and transfer the upper water-ethanol phase to a new tube.
8. Mix 0.1 ml of the upper phase with 0.7 ml distilled water, and dilute with 0.25% ethanol to prepare sample solution.

　
Extracellular SOD (EC-SOD)
1. Prepare a 0.5 ml volume of Con A-sepharose column equilibrated with PBS.
2. Apply supernatant of a tissue homogenate on the column and leave the column for 5 min at room temperature.
3. Add total 10 ml PBS to wash the column.
4. Add 1 ml of 0.5 M alpha-methylmannoside/PBS and collect the eluate. Repeat 5 times.
5. Use the eluate for the SOD assay without dilution. If the SOD activity is high enough, dilute the eluate with PBS.

　
Plant or Vegetable (200 mg)
1. Add 1 ml distilled water and homogenize the sample using a homogenizer with beads.
2. Filter the homogenate with paper filter, and lyophilize the filtrate.
3. Measure the weight of the lyophilized sample, and dissolve with 0.1 M phosphate buffer (pH 7.4) to prepare sample solution.

　
Tea(antioxidant activity detection)
1. Add 60 ml boiled water to 10 g tea and leave it for 2.5 min.
2. Filter the extract with paper filter and then filter again with 0.45 µm membrane filter.
3. Dilute the filtrate with distilled water to prepare sample solution.

　
Wine(antioxidant activity detection)
1. Filter wine with 0.45 µm membrane filter.
2. Dilute the filtrate with distilled water to prepare sample solution.